The long-term objective of this research plan is to develop a safe and effective immuocontraceptive for use by women. In previous studies, a completely synthetic chimeric peptide based on the sequence of sperm- specific lactate dehydrogenase (LDH-C4 epitope HC-5-19-TT) reversibly suppressed fertility in female baboons by 70 percent. In the current project period, we used sera from immunologically infertile patients to identify three new sperm-specific antigens which can be used alone or in combination with HC5-19-TT to increase the protection from pregnancy to greater than 90 percent. The immediate goal of this project is to test the safety and efficacy of a B-cell epitope from one of these antigens, the sperm-specific isoform of calpastatin (t-CAST), alone and in combination with the LDH-C4 epitope. Calpastatin is the peptide inhibitor of calpain, a cysteine protease that plays an important preteolytic role in vesicle exocytosis and membrane fusion events. Antibodies to t-CAST in the serum of an infertile patient (YM) agglutinated human sperm and inhibited sperm penetration of cervical mucus. A synthetic chimeric B-cell epitope of the protein (HTC25-41-TT) was immunogenic in rabbits and female macaques, and the anti-peptide sera agglutinated and immobilized human sperm. Therefore, t-CAST is present on the surface of acrosome intact sperm, and is a primary candidate for development of an immunocontraceptive. Four Specific Aims are proposed: 1) to establish the functional role of t-CAST during fertilization by targeted gene disruption in mice; 2) to establish contraceptive efficacy and reversibility of HTC25-41-TT in female cynomologus macaques; 3) to test a subunit vaccine composed of both HTC25-41-TT and HC5-19-TT in female macaques; and 4) to engineer E. coli to express large amounts of a recombinant form of the subunit vaccine for large-scale production under Good Laboratory Practices guidelines. This technology would provide a safe and effective alternative to presently available methods of contraception.